This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Here we test whether GM-CSF serves as an adjuvant for protection against a repeat low dose heterologous SIV challenge. Groups of 8 rhesus macaques were inoculated intramuscularly at 0 and 8 weeks with 3mg of SIV239 DNA, which did or did not co-express rhesus GM-CSF, and boosted with SIV239 MVA at 16 and 24 weeks. Both the DNA and MVA immunogens expressed Gag, PR, RT and Env. Twelve weekly low dose intrarectal challenges with 5000 TCID50 (1.8x107 copies of viral RNA) of SIV E660 were initiated 20 weeks following the final MVA inoculation. Protection against infection was plotted using the Kaplan-Meier method showing a nonparametric cumulative plot. The GM-CSF-adjuvanted immunizations increased protection against acquisition from 25% in the non adjuvanted group (6/8 infected) to 70% in the adjuvanted group (2/7 infected). Protection in the adjuvanted group was significantly different from protection of the controls (9/9 infected)(p=0.003). Co-expressed GM-CSF did not affect the titers of anti-Env binding Ab or the magnitudes or polyfunctionality of elicited CD4 and CD8 T cells. It did enhance the titers of neutralizing Ab for a neutralization sensitive variant of E660 (E660.11), and the avidity of anti-Env Ab for the 239 and E660 Envs. The prevention of infection showed a strong correlation with the avidity of the elicited Env-specific antibody for the Env of the SIVsmE660 challenge virus (r=0.9, p0.0001). These results demonstrate that GM-CSF expressed in cis can provide strong adjuvant activity for prevention of acquisition of a heterologous challenge by a SIV DNA/MVA vaccine.